Carboxylmethylation of various brain sources of Ca+2-calmodulin-dependent protein kinases decreased calmodulin-stimulated phosphorylation. This inhibition was noted in several cytosolic and membrane preparations of Ca+2-calmodulin kinase, and was also seen in purified preparations. Both tubulin and autophosphorylation patterns were inhibited in the purified enzyme. Analysis of carboxylmethylation revealed that the specific activity of methyl acceptor proteins increased from cytosol through partially purified preparations, with purified Ca+2-calmodulin kinase exhibiting the highest activity. Stoichiometry indicated that approximately 1.12 mol CH3/mol holoenzyme was incorporated in the purified enzyme. Also, acidic gel electrophoresis revealed that a protein of 62,000 daltons, which was common to all kinase preparations, was carboxylmethylated. This suggests that carboxylmethylation can modulate Ca+2-calmodulin dependent phosphorylation by directly carboxylmethylating the source of kinase activity.